Limits...
Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

Beyeler J, Schnyder I, Katsaros C, Chiquet M - PLoS ONE (2014)

Bottom Line: Wound closure rate showed highly significant differences between fibroblast strains.Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group.Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

Show MeSH

Related in: MedlinePlus

Fibroblasts from CLP patients fall into statistically distinct groups based on wound closure in vitro.Fibroblast strains were obtained from 16 CLP patients, 6 healthy individuals (foreskin; Fsk) and 3 patients with phimosis (Phim), and scratch wound assays were performed in vitro (see Materials and Methods). Box plots show the percentage of relative wound closure after 24 hours (% RWC) and the distribution of the collected data of at least three independent experiments (***p<0.001). (A) Box plots of the individual cell strains are marked at the bottom of the graph by the initials of the patients; they cluster into three distinct migratory groups namely “fast”, “intermediate” and “slow”. The origin of strains from the different proband groups is also indicated: Phimosis (Phim), cleft lip/and palate (CLP), normal foreskin (Fsk). See Tables S2 and S3 for descriptive data and p-values, respectively. (B) Representative images of one strain from each of the three migratory groups, taken immediately after wounding (0 h) and 24 hours later (24 h). Area of scale bar corresponds to 10% RWC or 0.32mm2. (C) Box plots of the median RWC values obtained for all cell strains within each of the three migratory groups. (D) Two distinct migratory groups within the CLP patient cohort are evident after performing a multiple comparisons test with the data collected from CLP fibroblast strains alone. The fibroblast strain derived from patient “BA” was considered an outlier and therefore not included in this statistical analysis. See Tables S2 and S4 for the descriptive data and p-values, respectively. (E) When CLP, Fsk and Phim cell strains were grouped separately, they corresponded to statistically distinct populations, although heterogeneity within the CLP group was evident from the large variance.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4216129&req=5

pone-0111752-g001: Fibroblasts from CLP patients fall into statistically distinct groups based on wound closure in vitro.Fibroblast strains were obtained from 16 CLP patients, 6 healthy individuals (foreskin; Fsk) and 3 patients with phimosis (Phim), and scratch wound assays were performed in vitro (see Materials and Methods). Box plots show the percentage of relative wound closure after 24 hours (% RWC) and the distribution of the collected data of at least three independent experiments (***p<0.001). (A) Box plots of the individual cell strains are marked at the bottom of the graph by the initials of the patients; they cluster into three distinct migratory groups namely “fast”, “intermediate” and “slow”. The origin of strains from the different proband groups is also indicated: Phimosis (Phim), cleft lip/and palate (CLP), normal foreskin (Fsk). See Tables S2 and S3 for descriptive data and p-values, respectively. (B) Representative images of one strain from each of the three migratory groups, taken immediately after wounding (0 h) and 24 hours later (24 h). Area of scale bar corresponds to 10% RWC or 0.32mm2. (C) Box plots of the median RWC values obtained for all cell strains within each of the three migratory groups. (D) Two distinct migratory groups within the CLP patient cohort are evident after performing a multiple comparisons test with the data collected from CLP fibroblast strains alone. The fibroblast strain derived from patient “BA” was considered an outlier and therefore not included in this statistical analysis. See Tables S2 and S4 for the descriptive data and p-values, respectively. (E) When CLP, Fsk and Phim cell strains were grouped separately, they corresponded to statistically distinct populations, although heterogeneity within the CLP group was evident from the large variance.

Mentions: Depending on the RWC, fibroblast strains could be divided into three migratory groups, namely “fast”, “intermediate”, and “slow” (Fig. 1A, B; Table S2), which were significantly different from one another. The p-values after multiple comparisons for the individual strains are listed in Table S3. The “fast” group included the 3 phimosis and 5 of the CLP strains. Their median RWC ranged between 41.8% and 46.4%. In the intermediate group, for which the RWC varied from 33.2% to 35.8%, 10 CLP and 3 normal foreskin strains were categorized. The slowest and smallest group (RWC = 26.1% –29.6%) was represented by 3 normal foreskin strains and 1 CLP “outlier” (strain “BA”). The migratory phenotype of each fibroblast strain within a group was stable when assays were repeated with different passages of cells from the same individual, as evidenced by the small variance of the combined measurements for individual strains (Fig. 1A). Statistically, these results were also confirmed by one-way ANOVA (p<2.2×10−16) followed by Tukey’s posthoc test using the means of each strain (“fast”, n = 8; “intermediate”, n = 13; “slow”, n = 4) for each migratory group (Fig. 1C; Table S2).


Accelerated wound closure in vitro by fibroblasts from a subgroup of cleft lip/palate patients: role of transforming growth factor-α.

Beyeler J, Schnyder I, Katsaros C, Chiquet M - PLoS ONE (2014)

Fibroblasts from CLP patients fall into statistically distinct groups based on wound closure in vitro.Fibroblast strains were obtained from 16 CLP patients, 6 healthy individuals (foreskin; Fsk) and 3 patients with phimosis (Phim), and scratch wound assays were performed in vitro (see Materials and Methods). Box plots show the percentage of relative wound closure after 24 hours (% RWC) and the distribution of the collected data of at least three independent experiments (***p<0.001). (A) Box plots of the individual cell strains are marked at the bottom of the graph by the initials of the patients; they cluster into three distinct migratory groups namely “fast”, “intermediate” and “slow”. The origin of strains from the different proband groups is also indicated: Phimosis (Phim), cleft lip/and palate (CLP), normal foreskin (Fsk). See Tables S2 and S3 for descriptive data and p-values, respectively. (B) Representative images of one strain from each of the three migratory groups, taken immediately after wounding (0 h) and 24 hours later (24 h). Area of scale bar corresponds to 10% RWC or 0.32mm2. (C) Box plots of the median RWC values obtained for all cell strains within each of the three migratory groups. (D) Two distinct migratory groups within the CLP patient cohort are evident after performing a multiple comparisons test with the data collected from CLP fibroblast strains alone. The fibroblast strain derived from patient “BA” was considered an outlier and therefore not included in this statistical analysis. See Tables S2 and S4 for the descriptive data and p-values, respectively. (E) When CLP, Fsk and Phim cell strains were grouped separately, they corresponded to statistically distinct populations, although heterogeneity within the CLP group was evident from the large variance.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216129&req=5

pone-0111752-g001: Fibroblasts from CLP patients fall into statistically distinct groups based on wound closure in vitro.Fibroblast strains were obtained from 16 CLP patients, 6 healthy individuals (foreskin; Fsk) and 3 patients with phimosis (Phim), and scratch wound assays were performed in vitro (see Materials and Methods). Box plots show the percentage of relative wound closure after 24 hours (% RWC) and the distribution of the collected data of at least three independent experiments (***p<0.001). (A) Box plots of the individual cell strains are marked at the bottom of the graph by the initials of the patients; they cluster into three distinct migratory groups namely “fast”, “intermediate” and “slow”. The origin of strains from the different proband groups is also indicated: Phimosis (Phim), cleft lip/and palate (CLP), normal foreskin (Fsk). See Tables S2 and S3 for descriptive data and p-values, respectively. (B) Representative images of one strain from each of the three migratory groups, taken immediately after wounding (0 h) and 24 hours later (24 h). Area of scale bar corresponds to 10% RWC or 0.32mm2. (C) Box plots of the median RWC values obtained for all cell strains within each of the three migratory groups. (D) Two distinct migratory groups within the CLP patient cohort are evident after performing a multiple comparisons test with the data collected from CLP fibroblast strains alone. The fibroblast strain derived from patient “BA” was considered an outlier and therefore not included in this statistical analysis. See Tables S2 and S4 for the descriptive data and p-values, respectively. (E) When CLP, Fsk and Phim cell strains were grouped separately, they corresponded to statistically distinct populations, although heterogeneity within the CLP group was evident from the large variance.
Mentions: Depending on the RWC, fibroblast strains could be divided into three migratory groups, namely “fast”, “intermediate”, and “slow” (Fig. 1A, B; Table S2), which were significantly different from one another. The p-values after multiple comparisons for the individual strains are listed in Table S3. The “fast” group included the 3 phimosis and 5 of the CLP strains. Their median RWC ranged between 41.8% and 46.4%. In the intermediate group, for which the RWC varied from 33.2% to 35.8%, 10 CLP and 3 normal foreskin strains were categorized. The slowest and smallest group (RWC = 26.1% –29.6%) was represented by 3 normal foreskin strains and 1 CLP “outlier” (strain “BA”). The migratory phenotype of each fibroblast strain within a group was stable when assays were repeated with different passages of cells from the same individual, as evidenced by the small variance of the combined measurements for individual strains (Fig. 1A). Statistically, these results were also confirmed by one-way ANOVA (p<2.2×10−16) followed by Tukey’s posthoc test using the means of each strain (“fast”, n = 8; “intermediate”, n = 13; “slow”, n = 4) for each migratory group (Fig. 1C; Table S2).

Bottom Line: Wound closure rate showed highly significant differences between fibroblast strains.Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group.Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthodontics and Dentofacial Orthopedics, School of Dental Medicine, University of Bern, Bern, Switzerland.

ABSTRACT
In a fraction of patients surgically treated for cleft lip/palate, excessive scarring disturbs maxillary growth and dento-alveolar development. Since certain genes are involved in craniofacial morphogenesis as well as tissue repair, a primary defect causing cleft lip/palate could lead to altered wound healing. We performed in vitro wound healing assays with primary lip fibroblasts from 16 cleft lip/palate patients. Nine foreskin fibroblast strains were included for comparison. Cells were grown to confluency and scratch wounds were applied; wound closure was monitored morphometrically over time. Wound closure rate showed highly significant differences between fibroblast strains. Statistically, fibroblast strains from the 25 individuals could be divided into three migratory groups, namely "fast", "intermediate", and "slow". Most cleft lip/palate fibroblasts were distributed between the "fast" (5 strains) and the "intermediate" group (10 strains). These phenotypes were stable over different cell passages from the same individual. Expression of genes involved in cleft lip/palate and wound repair was determined by quantitative PCR. Transforming growth factor-α mRNA was significantly up-regulated in the "fast" group. 5 ng/ml transforming growth factor-α added to the culture medium increased the wound closure rate of cleft lip/palate strains from the "intermediate" migratory group to the level of the "fast", but had no effect on the latter group. Conversely, antibody to transforming growth factor-α or a specific inhibitor of its receptor most effectively reduced the wound closure rate of "fast" cleft lip/palate strains. Thus, fibroblasts from a distinct subgroup of cleft lip/palate patients exhibit an increased migration rate into wounds in vitro, which is linked to higher transforming growth factor-α expression and attenuated by interfering with its signaling.

Show MeSH
Related in: MedlinePlus